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Vaccine. 2008 February 13; 26(7): 882–890.

Evaluation of the Langat/Dengue 4 Chimeric Virus as a Live

Attenuated Tick-Borne Encephalitis Vaccine for Safety and
Immunogenicity in Healthy Adult Volunteers

Peter F. Wright1, Sharon Ankrah1, Susan E. Henderson1, Anna P. Durbin2, Jim Speicher3,
Stephen S. Whitehead3, Brian R. Murphy3, and Alexander G. Pletnev3,*

1Department of Pediatrics, Division of Pediatric Infectious Disease, Vanderbilt University Medical Center,
Nashville, Tennessee 2Center for Immunization Research, Department of International Health, Johns Hopkins
Bloomberg School of Public Health, Baltimore, Maryland 3Laboratory of Infectious Diseases, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
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Abstract
With the steady rise in tick-borne encephalitis virus (TBEV) infections in Europe, development of
a live attenuated vaccine that will generate long-lasting immunity would be of considerable benefit.
A chimeric flavivirus, designated LGT/DEN4, was previously constructed to have a genome
containing the prM and E protein genes of Langat virus (LGT), a naturally attenuated member of the
TBEV complex, and the remaining genetic sequences derived from dengue 4 virus (DEN4). LGT/
DEN4 was highly attenuated in rodents and non-human primates, and clinical trials in humans were
initiated. Twenty-eight healthy seronegative adult volunteers were randomly assigned in a 4:1 ratio
to receive 103 PFU of LGT/DEN4 or placebo. Volunteers were closely monitored for clinical
responses and for blood chemistry and hematological changes, and the level of viremia and the
magnitude and duration of the neutralizing antibody response were determined. The LGT/DEN4
vaccine was safe and viremia was seen in only one vaccinee. Infection induced a neutralizing antibody
response to wild-type LGT in 80% of volunteers with a geometric mean titer (GMT) of 1:63 present
on day 42 post-immunization; however the antibody response against TBEV was both much less
frequent (35%) and lower in magnitude (GMT = 1:9). To assess the response to a booster dose, 21
of the original 28 volunteers were re-randomized to receive a second dose of either 103 PFU of
vaccine or placebo given 6 to 18 months after the first dose. The immunogenicity against either LGT
or TBEV was not significantly enhanced after the second dose of vaccine. Thus, chimerization of
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LGT with DEN4 yielded a vaccine virus that was highly attenuated yet infectious in humans. The
level of replication was sufficiently restricted to induce only a weak cross-reactive antibody response
to TBEV. To provide a sufficient level of immunity to widely prevalent, highly neurovirulent strains
of TBEV in humans, vaccine candidates will likely need to be based on the TBEV structural protein
genes.

*Corresponding author, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of
Health, 33 North Drive, Room 3W10A, MSC 3203, Bethesda, MD 20892-3203, USA. Phone # 301-402-7754, Fax # 301-480-0501, E-
mail: apletnev@niaid.nih.gov.
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 Wright et al. Page 2

Keywords
Tick-Borne Encephalitis; Live Virus Vaccine; Human
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1. Introduction
Tick-borne encephalitis (TBE) is a severe disease caused by antigenically closely related RNA
viruses belonging to the Flaviviridae family [1]. Members of the tick-borne encephalitis virus
(TBEV) antigenic complex, which includes Kyasanur forest disease, Central European and Far
Eastern tick-borne encephalitis, Langat, Louping ill, Omsk hemorrhagic fever, and Powassan
viruses, are transmitted in nature in a cycle between ticks and various mammalian species with
humans serving as incidental hosts. These viruses are endemic throughout most of the Northern
Hemisphere, and, except for Langat virus (LGT), cause human disease of varying severity with
up to 30% mortality [1]. Despite immunization of human populations living in endemic areas
with an inactivated virus vaccine, TBE remains a serious public health problem in Europe and
Asia, where up to 14,000 human cases are reported annually [2]. Licensed inactivated TBEV
vaccines are available in Europe and Russia that induce effective short-term protection;
however, three doses of vaccine are required for primary immunization. Since the titers of
neutralizing antibodies induced by the inactivated TBEV vaccine decline with time after
vaccination and with age, booster vaccinations every 3 years are needed to maintain protective
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immunity [3,4]. A TBEV vaccine that induces a more durable immunity would be an
improvement over the existing vaccine. Use of a live attenuated virus vaccine is the most likely
approach to achieve this goal since a single dose of the yellow fever (YF) 17D virus vaccine
provides relatively long-term immunity in humans [1].

Immunity to members of the TBEV complex is mediated in large part by neutralizing antibodies
directed to the structural envelope (E) glycoprotein of the virus. Since the amino acid identity
among the E glycoproteins of the TBEV complex of flaviviruses is 78% or greater [5–7], these
viruses share many protective E protein epitopes and hence form a single serogroup. Infection
or immunization with one member of the TBEV complex induces a moderate to highly cross-
reactive neutralizing antibody response to other members of the TBEV complex and confers
cross-resistance [8,9]. The E protein of LGT virus is 88% percent related in amino acid
sequence to that of strains of TBEV prevalent in Eurasia and is known to induce cross-
protective immunity to TBEV viruses [8,10,11]. However, the antibody response of infected
animals to LGT is higher than that to prevalent TBEV strains. It was hoped that the naturally
attenuated LGT virus could serve as a live attenuated virus vaccine candidate and induce a
protective and durable immune response to most members of the group [8,12–14]. Parenteral
immunization with live LGT virus induced immunity against TBEV, but unfortunately retained
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residual virulence for the CNS of humans [13]. Clearly, LGT would require further attenuation
before it could be used again as a vaccine candidate.

Modern recombinant DNA technology has made possible novel approaches for developing
live attenuated flavivirus vaccines (reviewed in [11] and [15]), and this technology has been
applied to further attenuate LGT for humans. The human trial reported here is part of the
development plan for a live attenuated TBEV vaccine [10,16–20] that is based on chimerization
of a non-neuroinvasive, mosquito-borne dengue type 4 virus (DEN4) with a neurotropic, tick-
borne LGT virus. The chimeric virus tested here is referred to as LGT/DEN4. Specifically,
chimeric LGT/DEN4 virus was created by replacing the membrane precursor (prM) and
envelope (E) structural protein genes of DEN4 with the corresponding genes from LGT strain
TP21 [18,20]. The resulting LGT/DEN4 virus exhibited greatly reduced neuroinvasiveness
and neurovirulence in mice compared to the LGT parent and was immunogenic, providing
complete protection against lethal challenge with TBEV or LGT [18–20]. LGT/DEN4 was also

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found to be attenuated, immunogenic, and efficacious in monkeys [10,20]. In addition, the

LGT/DEN4 virus failed to infect mosquitoes after intrathoracic inoculation [10], indicating
that the chimeric virus has a limited potential for transmission by mosquitoes. Based on these
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preclinical studies, LGT/DEN4 was selected as the initial TBEV vaccine candidate for
evaluation in a clinical trial in humans at a dose of 103 PFU, since this dose of vaccine candidate
was able to induce protective immunity against LGT or TBEV in mice and monkeys [10,18–
20]. Similarly attenuated vaccine constructs have been generated using a Far Eastern TBEV
strain as the tick-borne virus parent [16,20].

Two major questions were addressed in the present study of LGT/DEN4 in adult volunteers.
First, did chimerization between the genetically unmodified LGT and DEN4 viruses lead to
attenuation in humans as it did in mice and monkeys? Second, did the level of antibody induced
by the attenuated infection with LGT/DEN4 in humans stimulate a sufficient level of cross-
reactive antibodies to pathogenic Eurasian TBEV strains to warrant continued development?
The findings indicate that chimerization indeed led to attenuation of the LGT/DEN4 vaccine
candidate for humans as it did for monkeys. However, the reduced level of LGT/DEN4
replication resulted in the induction of a low titer of antibodies against TBEV.

2. Materials and methods

2.1. Study population
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The clinical phase 1 study was conducted in the General Clinical Research Center at Vanderbilt
University Medical Center (supported by GCRC grant M01RR00095). Laboratory assays were
performed at Johns Hopkins School of Public Health and the Laboratory of Infectious Diseases
at NIH. The clinical protocol was reviewed and approved by the Vanderbilt University
Institutional Review Board, the Vanderbilt University Biosafety Committee, the Committee
on Human Research of the Bloomberg School of Public Health, and the Johns Hopkins
University Institutional Biosafety Committee. A medical monitor, Dr. Robin McKenzie and
the NIAID Data Safety and Monitoring Board reviewed the study at predetermined intervals.

Twenty-eight healthy adult volunteers were recruited from the Nashville area. Informed
consent was obtained from each volunteer in accordance with the Code of Federal Regulations
Title 21, Part 50 – Protection of Human Subjects. The individuals who were enrolled met the
following eligibility criteria: age 18–50 years; no history of chronic illness; normal findings
during physical examination; negative (<1:10) for serum neutralizing antibodies to LGT,
TBEV, West Nile virus, dengue virus types 1–4, and yellow fever virus; negative for antibodies
to Saint Louis encephalitis virus and Japanese encephalitis virus by hemagglutination-
inhibition (titer <1:10); negative human immunodeficiency virus antibody test and hepatitis C
virus antibody test; negative for hepatitis B surface antigen; normal hematologic and hepatic
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values; and normal urinalysis. Female volunteers had a negative result on a urine pregnancy
test prior to vaccinations and on the days of vaccination and agreed to use contraception or
abstain from sexual intercourse for the duration of the study.

2.2. Study design

The initial phase of the study was a double-blind placebo-controlled trial in which volunteers
were randomly assigned in a 4:1 ratio to receive the LGT/DEN4 vaccine or placebo. Twenty-
eight volunteers were enrolled in the study; 20 received 103 plaque forming units (PFU) of the
LGT/DEN4 vaccine and eight received placebo consisting of vaccine diluent (L-15 Medium
without phenol red; Cambrex Bio Science Walkersville, Inc., Rockville, MD). Volunteers were
enrolled in a step-wise manner: four volunteers were initially enrolled; after study day 21, all
safety data was reviewed by the medical monitor before proceeding with enrollment of the
next 10 volunteers; and the set of safety data collected for these volunteers was then reviewed

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by the medical monitor before enrollment of the remaining 14 volunteers. Randomization was
done such that no more than 1 volunteer in the first group of 4 volunteers and no more than 4
volunteers in the first group of 10 volunteers received placebo.
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Each volunteer received a single 0.5 ml dose of vaccine or placebo as a subcutaneous injection
in the deltoid region. Volunteers were observed 30 minutes after vaccination for any immediate
reaction to the vaccine. They were given a diary card and a digital oral thermometer to record
their temperature 3 times daily for 19 days. Volunteers returned to the clinic every other day
for 16 days after vaccination and on study days 19, 21, 28, 42, and 180. A clinical assessment
was done at each visit and blood was obtained for hematological and clinical chemistry testing
and for virologic or immunologic analysis. Serum from study days 0, 2, 4, 6, 8, 10, 12, 14, and
16 was titered for vaccine virus as described below; serum from study days 0, 28, 42, and 180
was analyzed for neutralizing antibody.

After completion of initial vaccination, the study was modified to include a second dose to
assess the response of vaccinees to a booster dose. Twenty-one of the original 28 volunteers
agreed to participate. Six to 18 months after initial vaccination they were re-randomized to
receive either 103 PFU of vaccine or the placebo control (vaccine diluent). Sixteen volunteers
received vaccine as the primary and secondary dose, 3 placebo recipients from the primary
phase received vaccine in the booster phase, while 2 volunteers received placebo in both study
phases.
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2.3. Adverse Event Monitoring

Local reactogenicity was defined as injection site erythema, tenderness or swelling. Erythema
and swelling at the injection site were graded as mild (1 - ≤ 20 mm diameter), moderate (>20mm
- <50 mm diameter), or severe (≥50 mm diameter). Clinical signs and symptoms such as fever,
headache, photophobia, nuchal rigidity, mental status change, nausea, malaise, myalgia,
arthralgia, and rash were assessed at each follow-up visit during the acute 19-day post-
vaccination period. Injection site pain and other solicited adverse events were graded as mild
(no effect on activities of daily living, no treatment given); moderate (partial limitation in
activities of daily living or treatment given); or severe (activities of daily living limited to <50%
of baseline or medical evaluation required). A serious adverse event was defined as any event
resulting in a life-threatening condition, hospitalization, congenital anomaly or birth-defect,
persistent or significant disability, or death. All adverse events were determined to be either
definitely, probably, possibly, remotely, or not related to vaccine. A vaccine-related
meningoencephalitic-like syndrome was defined as infection associated with 2 or more of the
following symptoms: grade 2 headache lasting ≥ 12 hours, grade 2 photophobia lasting ≥ 12
hours, nuchal rigidity (if present is grade 3, by definition), or generalized myalgia, grade 2,
lasting ≥ 12 hours. LGT/DEN4 virus infection was defined as recovery of vaccine virus from
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the serum of a volunteer or a ≥4-fold rise in titer of serum neutralizing antibodies against LGT.

2.4. Laboratory testing

Clinical laboratory studies done for safety assessment included the following: a complete blood
count including a white cell differential was tested at every visit for the first 19 days of the
trial; serum alanine amino transferase (ALT) was tested at every follow-up visit from day 4
through day 19 and again on study day 28; prothrombin time, partial thromboplastin time,
serum creatinine, and creatine phosphokinase were tested at every other visit through study
day 16 and again on study day 28. The above studies were performed in the Vanderbilt Medical
Center Clinical Pathology Laboratories. Serum from each visit through study day 19 was titered
for vaccine virus and, if positive on study day 19, serum from study day 21 was also titered.
Virus titer was determined at the Center for Immunization Research Laboratory, Bloomberg
School of Public Health (Baltimore, MD).

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2.5. Vaccine virus

A full-length cDNA copy of LGT/DEN4 in which the prM and E structural protein genes of
DEN4 were replaced with those of LGT strain TP21 was constructed as previously described
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[18,20]. Infectious RNA transcripts derived from the chimeric LGT/DEN4 cDNA were used
to transfect qualified mosquito C6/36 cells. Recovered virus was passaged once in qualified
Vero cells, terminally diluted twice and then amplified in Vero cells. The final amplification
of vaccine seed virus was made in serum-free medium. The clinical vaccine lot was produced
at Novavax (Rockville, MD) in compliance with Good Manufacturing Practices and passed all
safety tests confirming microbial sterility and animal safety. The LGT/DEN4 vaccine lot
(designated LGT TP21/DEN4#2) had a titer of 4 × 106 PFU/ml. Vaccine virus in OptiPRO
serum-free medium (Invitrogen, Carlsbad, CA) containing SPG buffer (final concentration:
218 mM sucrose, 5.4 mM monosodium glutamate, 3.8 mM potassium phosphate, monobasic
and 7.2 mM potassium phosphate, dibasic, pH 7.2) was diluted with sterile L-15 medium to
yield 103 PFU/0.5 ml just prior to vaccination and was kept on wet ice until administration.
The titer of the LGT/DEN4 vaccine virus at the time of administration was confirmed by
titration on Vero cell monolayer cultures as described previously [10]. Titration of the vaccine
virus was done immediately after preparation and then 4 hours after preparation to assure
stability throughout the vaccination period. Back titrations of the LGT/DEN4 inocula indicate
that vaccine virus was stable during the time of administration, with its titers ranging from
102.6 to 102.9 PFU/dose.
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2.6. Virus quantitation and serologic assessment

The amount of virus in blood was determined by plaque-forming assay after inoculation of
serial 10-fold dilutions of serum onto Vero cell monolayer cultures as described previously
[10]. LGT- or TBEV-specific neutralizing antibody titer of each serum sample was determined
by a plaque-reduction neutralizing assay using LGT TP21 or TBEV/DEN4Δ30 virus,
respectively [20]. A chimeric TBEV/DEN4Δ30 virus contained the prM and E protein genes
of the highly virulent Sofjin strain of Far Eastern TBEV. The E protein of Sofjin strain shares
94–96% of amino acid identity with TBEV strains endemic in Europe and Asia [21].
Neutralizing antibody titer was defined as the dilution of serum that neutralized 60% of virus
used in the assay. Serum of rhesus monkeys previously immunized with LGT/DEN4 or TBEV/
DEN4Δ30 virus and then challenged with wild-type LGT virus [20] was used for positive
control.

The serological responses were quantified as neutralizing antibody titers and classified in two
ways: subjects with detectable antibody (≥1:5) and subjects with a fourfold rise in antibody
titer (≥1:20). The former category was enumerated separately since the day 0 serum specimens
of all subjects and all post-immunization specimens in placebo recipients had titers of <1:5,
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suggesting that vaccinees with detectable antibody had a response to vaccine. Such serum
specimens are referred to as seropositive. Vaccinees with titers of ≥1:20 were considered
unequivocally infected with vaccine, and vaccinees with this level of antibody were considered
to have seroconverted.

3. Results
3.1. Volunteers
Twenty-eight volunteers ranging in age from 18 to 50 years were enrolled in the initial phase
of the study and followed for the next 6 months. Seventeen volunteers were male (100% white)
and 11 were female (64% white, 27% black and 9% Asian). Twenty received vaccine and eight
received placebo.

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Of the 21 volunteers in the booster dose phase of the study, 14 were male and 7 were female
(6 white and 1 Asian). Nineteen volunteers (12 male and 7 female) received vaccine while 2
volunteers, both male, received placebo.
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3.2. Clinical Safety

3.2.1. Initial dose—Vaccine was well tolerated by volunteers with minimal local
reactogenicity (2/20 vaccinees had mild tenderness at the injection site, 2 additional vaccinees
had bruising at the injection site, and 1/10 placebo recipients had mild arm stiffness related to
the injections). None of the volunteers experienced any signs or symptoms of systemic or
meningoencephalitic-like illness and there were no severe reactions (Table 1). Of the 20 LGT/
DEN4 vaccinees, 5 reported headache, a frequency similar to that reported by 3 of 8 placebo
recipients. One vaccinee and one placebo recipient developed neutropenia, and both cases
resolved uneventfully. One placebo recipient reported a fever of 101.5°F; no temperature
elevations were reported by vaccinees.

3.2.2. Booster dose—All 19 volunteers in the booster phase of the study tolerated the LGT/
DEN4 vaccine well (Table 1). Two vaccinees had mild local tenderness at the injection site.
Three vaccinees reported having headaches that were judged possibly related to the vaccine.
All the headaches were classified as not serious and the longest duration was 42 hours. Rash
was not reported or observed in any of the volunteers during this phase of the study.
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3.3. Evidence of viremia

One vaccinee (subject #25 in Table 3) had vaccine virus detected in the blood following
vaccination. This viremia occurred during the booster dose phase of the trial in a vaccinee who
had received placebo at first vaccination. Vaccine virus was recovered on study day 8 at the
lowest detectable level (titer = 0.7 log10 PFU/ml). The low level of viremia in infected
vaccinees indicated that LGT/DEN4 virus was highly restricted in replication in humans.

3.4. Serologic responses to LGT/DEN4

Ninety-five percent (19/20) of vaccinees were seropositive to LGT as tested on study days 28,
42, or 180 (Table 2), but only 80% were considered to have seroconverted. Geometric mean
titers (GMT) of neutralizing antibody to LGT were highest on day 28 and 42 (66 and 63
reciprocal GMT, respectively) with about a three-fold drop in GMT by study day 180. By using
an attenuated TBEV/DEN4Δ30 virus as the test virus in the neutralization assay, TBEV-
specific cross-reactive antibodies in the serum of volunteers could be measured following
vaccination with LGT/DEN4. Of the 20 volunteers, 13 (65%) had detectable TBEV-specific
neutralizing antibody titers (≥1:5) on study days 28, 42, or 180, but antibody titers to
heterologous TBEV were much less than to the homologous LGT virus (Table 2). Overall,
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only 35% of vaccinees seroconverted to TBEV. Antibody was not detected against either LGT
or TBEV in any placebo recipient.

All volunteers who received two doses of LGT/DEN4 administered with an interval of at least
6 months after primary inoculation became seropositive (≥1:5) against LGT (Table 3).
Geometric mean LGT-specific neutralizing antibody titers in the serum of vaccinees were
similar on day 28, 42, or 180 between groups that received one or two doses of vaccine. In
both groups, antibody titers peaked at day 28 or 42 and tended to decrease about two-fold by
day 180. Two subjects (#17 and 28) in the booster group who did not seroconvert following
primary inoculation seroconverted 28 days after receiving the booster inoculation, however,
the antibody response of one of them (#28) became undetectable again by day 180. The second
dose of LGT/DEN4 failed to significantly increase the number of vaccinees with antibody to
TBEV (6/16 before the second dose to 8/16 after second dose) or to increase the mean titer of
TBEV antibody.

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4. Discussion
Experience with an extensive vaccination campaign against TBEV in Austria during the last
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20 years has shown that active immunization of human populations living or traveling in
endemic areas can result in a decline of illness caused by TBEV [22]. The currently available
TBEV vaccines licensed in Europe (FSME-IMMUN® vaccine in Austria and Encepur®
vaccine in Germany) are derived from inactivated whole virus (Central European TBEV strain
Neudoerfl and strain K23, respectively). Although each vaccine is administered on a slightly
different schedule, three immunizations are recommended: on day 0 (1st dose), from days 21
to 72 (2nd dose), and after 9–12 months (3rd dose) [23]. These licensed vaccines are safe, well
tolerated by both adults and children, and immunogenic with seroconversion rates of 90–99%
following the primary immunization course [22,23]. For these vaccines, seroconversion is
defined as a change from an undetectable immunological response (titer <1:2) to a detectable
level (titer ≥1:2) in the neutralization assay. However, titers of neutralizing antibodies wane
in a log-linear fashion within 2–4 years after vaccination, falling below protective levels
(neutralizing antibody titer of <1:10) in 5–30% of the recipients [3]. The decrease in post-
vaccination antibody levels is observed with increased frequency in the elderly, such that
approximately 30% of persons over 60 years of age do not have protective immunity. Currently,
booster vaccinations are recommended every 3 years after completion of primary
immunization.
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Despite the effectiveness of TBE vaccination in Austria, the inactivated vaccines have several
limitations, including the long schedule of primary immunization, the need for repeated booster
vaccinations due to the short duration of immunity, the rare occurrence of severe TBE disease
in vaccinees in TBEV endemic areas [4,24], and the high cost of manufacture. These
disadvantages and difficulties associated with the current inactivated TBEV vaccines have
encouraged research to develop new improved vaccines. Based on the success of live attenuated
vaccines for other flaviviruses, such as the yellow fever 17D vaccine [25] and Japanese
encephalitis (JE) SA 14-14-2 vaccine [26], live attenuated vaccines offer the possibility of
inducing the desired protective and durable immune responses. Early attempts to develop an
effective live attenuated TBEV vaccine using the naturally attenuated LGT strains were
unsatisfactory due to a low frequency of post-vaccination encephalitis [11,13,27].
Nevertheless, these vaccine candidates induced a broad cross-protective immune response
against TBEV strains that lasted 3 or more years following a single immunization [8,12,13,
27]. The LGT virus vaccine candidate tested in Russia was effective in preventing TBE in
endemic regions, reducing the incidence of TBE by more than 10-fold compared to the
previously used inactivated TBEV vaccine (reviewed in [11] and [13]).

The pre-clinical studies with LGT/DEN4 indicated that chimerization of LGT with DEN4
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strongly attenuated the resulting chimeric virus for rodents and non-human primates.
Attenuation was manifest as reduced replication in human cells of neural origin, decreased
neurovirulence in newborn mice as measured by intracerebral inoculation, and decreased
neuroinvasiveness in immunocompetent or immunodeficient mice, a property that reflects the
capacity of virus to spread from a peripheral site of the body into the CNS where it causes
encephalitis [20]. In addition, LGT/DEN4 was found to be highly restricted in replication in
rhesus monkeys, replicating to levels significantly less than either parent virus [10], indicating
that chimerization itself led to an increased level of attenuation. It is likely that the presence
of the LGT prM and E genes in the chimeric virus creates LGT-DEN4 protein incompatibilities
that compromise virus replication in vivo. Although chimerization between LGT and DEN4
indeed resulted in an acceptable level of attenuation for monkeys, chimerization between two
flaviviruses does not always lead to a satisfactory level of attenuation. YF/JE-N [28,29], St.
Louis encephalitis/DEN4 (A.G. Pletnev; unpublished data), intertypic DEN1/DEN4 [30], or
intertypic JE virus chimeras [31] do not appear to be satisfactorily attenuated in mice or

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monkeys. All chimeric viruses evaluated to date involved a parent virus that contained one or
more attenuating mutations [32,33]. Since the current human study was the first to evaluate a
chimeric flavivirus generated from two unmodified parent viruses, this permitted an evaluation
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of the independent effect of chimerization on virus attenuation for humans.

The LGT/DEN4 vaccine candidate was highly attenuated for humans following either the first
or second dose without significant local or systemic reactogenicity. The clinical reactogenicity
observed in recipients of the LGT/DEN4 vaccine was less than that observed in recipients of
a similar dose of a previously reported live attenuated dengue DEN4 vaccine [32]. Rash and
neutropenia were observed in some recipients of the previously tested DEN4 vaccine candidate,
but not in LGT/DEN4 vaccinees. Additional evidence of the further attenuation of this vaccine
came from the observation that only one of 17 vaccinees infected with LGT/DEN4 (those with
at least a 4-fold rise in antibody titer) had detectable viremia. Since dengue viruses replicate
in humans up to 106–8 infectious units per ml blood [34] and since previous studies in humans
with LGT TP21 virus revealed that over 70% of volunteers became viremic for a duration
ranging from 3 to 10 days [27], it is clear that chimerization of LGT with DEN4 greatly
decreased the replication of the chimeric virus in humans compared to that of either parent.
The level of replication of LGT/DEN4 virus in humans was similar to that observed in rhesus
monkeys [20], indicating that chimerization resulted in attenuation in both species. This
interpretation is offered with the caveat that the replication of the wild-type DEN4 parent has
not been evaluated directly in humans. However, LGT/DEN4 induced viremia much less
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frequently in healthy adult volunteers than that observed for live attenuated DEN4Δ30 vaccine
candidate derived from the same DEN4 parent and administered at the same dose [32].

A single 103 PFU dose of LGT/DEN4 vaccine candidate induced a moderate neutralizing
antibody response against LGT: 95% of volunteers became seropositive post-vaccination (titer
≥1:5) and 80% seroconverted as defined by a ≥4-fold increase in serum neutralizing antibody
titer (titer ≥1:20). However, the antibody response against heterologous TBEV was
unacceptably low in terms of both the frequency and the magnitude of the response. The levels
of antibody to TBEV achieved in LGT/DEN4-immunized volunteers appeared less than that
observed in recipients of the licensed inactivated TBEV vaccines described above. Two
independent factors may have contributed to this weak heterologous TBEV antibody response:
(1) the 12% divergence in amino acid sequence of the structural E proteins of LGT and the Far
Eastern strain of TBEV [35] and (2) the low level of replication of the LGT/DEN4 virus in
humans. The observation that almost all vaccinees were without detectable viremia and did
not develop a strong neutralizing antibody response to the homologous LGT virus suggests
that LGT/DEN4 was highly attenuated for humans. Clearly the additional restriction of
replication resulting from the chimerization changed the LGT virus from an immunogenic
vaccine candidate to one that is over-attenuated.
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Interestingly, the neutralizing antibody response against either LGT or TBEV was not boosted
after the second dose of LGT/DEN4 vaccine, suggesting that the relatively low level of
immunity associated with a single dose of LGT/DEN4 immunization was able to restrict the
virus replication to levels insufficient to stimulate an anamnestic response. In contrast, booster
antibody responses are frequently observed in monkeys after second doses of dengue virus
[36]. Perhaps the secondary immune response to the encephalitic flaviviruses is fundamentally
different from that of the dengue viruses, and resolution of this interesting question will require
additional clinical trials.

The failure to induce a vigorous immune response against TBEV in humans using the LGT/
DEN4 chimeric virus confirms that the required balance between attenuation and
immunogenicity of live virus vaccines is difficult to achieve. It may be possible to overcome
the low level of immunogenicity against TBEV by increasing the dose. However, previous

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studies with other flavivirus vaccine candidates in rhesus monkeys and humans have shown
that the magnitude of the immune response is not necessarily a function of the vaccine dose
[10,32]. Therefore, we believe that achieving >95% seroconversion to TBEV while
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maintaining a satisfactory safety profile will be difficult to accomplish by increasing the dose
of immunization. As an alternative strategy, our current live attenuated TBEV vaccine
candidate is the chimeric TBEV/DEN4Δ30 virus that is highly attenuated in mice and rhesus
monkeys and induces a higher level of neutralizing antibodies in the monkeys against TBEV
than did LGT/DEN4. The structural proteins of the TBEV/DEN4Δ30 chimera are antigenically
closely related (94–96% of amino acid identity) to the TBEV strains that are responsible for
causing most TBEV illness in Eurasia. We anticipate that the TBEV/DEN4Δ30 chimera will
induce a more satisfactory immune response against TBEV in humans than that observed in
the present study using divergent E protein of the LGT/DEN4 chimera. However, its safety in
primates will require additional studies.

In summary, LGT/DEN4 vaccine virus administered as a single 103 PFU dose was highly
infectious and highly attenuated in human volunteers and induced a neutralizing antibody
response to LGT in at least 80% of recipients. However, seroconversion against heterologous
TBEV was infrequent and the level of TBEV antibodies was significantly lower in magnitude
than that observed against homologous LGT virus. The neutralizing antibody titers against
either LGT or TBEV were not significantly increased after a second dose of LGT/DEN4
vaccine indicating that an immunization with a single dose of vaccine was sufficient to restrict
NIH-PA Author Manuscript

virus replication and blunt an anamnestic response. Nevertheless, the second dose was safe
with no evidence of enhanced replication or potentiation of disease.

Acknowledgements
This research was supported in part by the Intramural Research Program of the NIH, NIAID and by grant M01RR00095
of the General Clinical Research Center at Vanderbilt University Medical Center.

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Table 1
Clinical summary of volunteers inoculated with a primary or booster dose of LGT/DEN4, a live attenuated vaccine candidate for TBE.

Volunteers given Dose cohort No. of volunteers No. of viremic Number of volunteers with indicated sign:
(log10PFU)
Systemic illnessa Headache Rash Fever Neutropeniac Elevated ALTd

Initial LGT/DEN4 3.0 20 0 0 5 0 0 1 0

Wright et al.

Initial placebo 8 0 0 3 0 1b 1 0
Boost LGT/DEN4 3.0 19 1 0 3 0 0 0 0
Boost placebo 2 0 0 0 0 0 0 0

a
Systemic illness is defined as ≥2 of the following symptoms lasting ≥2 days: headache, malaise, vomiting, arthralgia/malaise, nausea, or photophobia. There was no significant difference between
vaccines and placebo recipients in the occurrence of any of the individual solicited symptoms used to define systemic illness. All solicited adverse events were mild or moderate in severity.
b
Fever occurred on study day 40; maximum temperature was 101.5°F.
c
Neutropenia is defined as an absolute neutrophil count of <1500 cells/mm3.
d
Elevated ALT level is defined as any value above the upper limit of normal (for males, >72U/L; for females, >52 U/L).

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Table 2
Immunologic response of volunteers immunized with a single 103 PFU dose of the chimeric LGT/DEN4 vaccine candidate.

Volunteer ID# Code Serum neutralizing antibody titer on indicated day againsta:

TBEV/DEN4Δ30 LGT

0 28 42 180 0 28 42 180
Wright et al.

02 Placebo <5 <5 <5 NTb <5 <5 <5 <5

06 Placebo <5 <5 <5 <5 <5 <5 <5 <5
12 Placebo <5 <5 <5 <5 <5 <5 <5 <5
14 Placebo <5 <5 <5 <5 <5 <5 <5 <5
16 Placebo <5 <5 <5 <5 <5 <5 <5 <5
19 Placebo <5 <5 <5 <5 <5 <5 <5 <5
25 Placebo <5 <5 <5 <5 <5 <5 <5 <5
26 Placebo <5 <5 <5 <5 <5 <5 <5 <5

01 Vaccine <5 14 9 <5 <5 300 404 101

03 Vaccine <5 <5 <5 12 <5 10 20 <5
04 Vaccine <5 23 8 33 <5 210 97 175
05 Vaccine <5 318 164 186 <5 3005 1702 283
08 Vaccine <5 9 6 18 <5 43 27 41
09 Vaccine <5 <5 7 10 <5 35 25 11
10 Vaccine <5 <5 <5 <5 <5 18 11 5
11 Vaccine <5 11 8 25 <5 250 119 25
13 Vaccine <5 <5 <5 <5 <5 41 26 30
15 Vaccine <5 <5 <5 27 <5 54 64 65
17 Vaccine <5 <5 <5 <5 <5 <5 <5 15
18 Vaccine <5 <5 <5 <5 <5 90 75 <5
20 Vaccine <5 <5 <5 <5 <5 <5 <5 <5
21 Vaccine <5 5 5 NT <5 88 119 NT
22 Vaccine <5 <5 <5 <5 <5 67 47 <5
23 Vaccine <5 21 304 276 <5 145 833 217
24 Vaccine <5 30 48 175 <5 264 516 45
27 Vaccine <5 8 5 9 <5 55 33 8
28 Vaccine <5 <5 <5 6 <5 <5 6 <5
29 Vaccine <5 16 25 131 <5 1252 850 191

GMTc: 9 9 16 66 63 24

Vaccine. Author manuscript; available in PMC 2009 February 13.

Seropositive: 10/20 11/20 12/19 17/20 18/20 14/19
50% 55% 63% 85% 90% 74%

Seroconversion: 4/20 4/20 7/19 15/20 16/20 10/19

20% 20% 37% 75% 80% 53%
7/20 = 35% 16/20 = 80%

a
Plaque reduction (60%) neutralizing antibody titers (reciprocal) were determined against wild-type LGT TP21 strain or chimeric TBEV/DEN4Δ30 virus as indicated.
b
NT, not tested.
c
GMT, geometric mean titers (reciprocal) are calculated for all 20 vaccine recipients. For calculation of GMT, a negative titer (<1:5) was assigned a value of 4. Seroconversion (indicated by shading) is
defined as a serum neutralizing antibody titer of ≥1:20 on study day 28, 42, or 180, compared with the pre-vaccination titer, which was <1:5 for each volunteer.
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Table 3
Immunologic response of volunteers following a second 103 PFU dose of the chimeric LGT/DEN4 vaccine candidate.

Volunteer ID# Code Serum neutralizing antibody titer on indicated day againsta:

TBEV/DEN4Δ30 LGT

1-st dose 2-nd dose 0 28 42 180 0 28 42 180

Wright et al.

19 Placebo Placebo <5 <5 <5 <5 <5 <5 <5 <5
26 Placebo Placebo <5 <5 <5 <5 <5 <5 <5 <5

12 Placebo Vaccine <5 <5 <5 <5 <5 <5 <5 <5
16 Placebo Vaccine <5 <5 <5 <5 <5 <5 <5 <5
25 Placebo Vaccine <5 298 568 68 <5 1008 649 147

01 Vaccine Vaccine <5 6 5 <5 19 39 22 28

03 Vaccine Vaccine <5 <5 <5 <5 <5 17 11 5
04 Vaccine Vaccine 38 36 37 36 157 187 164 83
05 Vaccine Vaccine 39 51 31 28 289 446 381 86
08 Vaccine Vaccine 14 39 33 28 28 100 88 26
10 Vaccine Vaccine <5 <5 <5 <5 <5 6 5 7
11 Vaccine Vaccine 8 9 5 37 34 48 52 56
13 Vaccine Vaccine <5 <5 <5 <5 14 29 31 20
17 Vaccine Vaccine <5 <5 <5 <5 <5 58 92 30
18 Vaccine Vaccine <5 <5 <5 <5 11 21 14 13
20 Vaccine Vaccine <5 <5 <5 <5 <5 16 14 <5
22 Vaccine Vaccine <5 <5 <5 <5 10 36 44 11
23 Vaccine Vaccine 35 30 76 10 55 85 67 49
27 Vaccine Vaccine <5 <5 5 <5 5 11 12 7
28 Vaccine Vaccine <5 7 <5 <5 <5 50 19 <5
29 Vaccine Vaccine 17 11 9 14 127 163 164 80

GMTb: 8 9 8 8 17 45 38 19

Seropositive: 6/16 8/16 8/16 6/16 11/16 16/16 16/16 14/16

38% 50% 50% 38% 69% 100% 100% 88%

Seroconversion: 0/16 0/16 1/16 2/16 2/16 1/16

0% 0% 6% 13% 13% 6%
1/16 = 6% 3/16 = 19%

Vaccine. Author manuscript; available in PMC 2009 February 13.

a
Plaque reduction (60%) neutralizing antibody titers (reciprocal) were determined against wild-type LGT TP21 strain or chimeric TBEV/DEN4Δ30 virus as indicated.
b
GMT, geometric mean titers (reciprocal) are calculated for 16 vaccine recipients and does not include 3 volunteers who received placebo in primary inoculation and vaccine in second inoculation.
Seroconversion (indicated by shading) is defined as a ≥4-fold rise in serum neutralizing antibody titer on day 28, 42, or 180 after booster vaccination, compared with a titer observed in serum of volunteers
prior to receive a booster dose of LGT/DEN4 vaccine.
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